As is well known in the field of life sciences, tandem mass spectrometry (MS/MS) is a powerful tool for structural elucidation of analytes, and in its many permutations, MS/MS is commonly used to dissociate and analyze such diverse species as peptides, proteins, small molecule drug compounds, synthetic polymers, and metabolites. The most common method of causing ion fragmentation in MS/MS analyses is collision induced dissociation (CID), in which a population of analyte precursor ions are accelerated into target neutral gas molecules such as nitrogen (N2) or argon (Ar), causing the precursors to gain internal energy and fragment. The ionic fragments ions are analyzed so as to provide useful information regarding the structure of the precursor ion.
When performing MS/MS in an ion trap, there are various ways to activate ions in order to cause ion fragmentation by means of collision induced dissociation or otherwise. The most efficient and widely used method involves collision-induced dissociation by means of a resonance excitation process. This method, which may be referred to as RE-CID, utilizes an auxiliary alternating current voltage (AC) that is applied to the ion trap in addition to the main RF trapping voltage. This auxiliary voltage typically has relatively low amplitude (on the order of 1 Volt (V)) and duration on the order of tens of milliseconds. The frequency of this auxiliary voltage is chosen to match an ion's frequency of motion, which in turn is determined by the main trapping field amplitude and the ion's mass-to-charge ratio (m/z). As a consequence of the ion's motion being in resonance with the applied voltage, the ion takes up energy from this voltage, and its amplitude of motion grows.
FIG. 1A schematically illustrates a resonant excitation process, using a quadrupole ion trap as an example. In FIG. 1A, a quadrupole ion trap 100 comprises a ring electrode 102 and end cap electrodes 104a, 104b, as is known in the art. Without application of a supplementary AC voltage, the oscillating RF quadrupole field generated within the trap 100 causes an ion 106 to remain trapped with a certain kinetic energy state represented by the dashed arrow. In this particular energy state, the kinetic energy of the ion 106 is generally insufficient to cause fragmentation of the ion during occasional collisions with molecules 108 of an inert bath gas. If a supplementary resonance voltage of the proper frequency is subsequently continuously applied, then, in an ideal quadrupole field, the ion's amplitude will grow linearly with time, as is indicated by the solid spiral arrow in FIG. 1A. The ion's kinetic energy increases with the square of the ion's amplitude of motion and, therefore any collisions which occur with neutral gas molecules (or other ions) become increasingly energetic. At some point during this process, the collisions which occur deposit enough energy into the molecular bonds of the ion in order to cause those bonds to break, and the ion to fragment. As one example, the ion 106 may fragment into a smaller ion 110 and a neutral molecule 112.
A variant of the CID technique, referred to as pulsed-q dissociation (PQD or, alternatively, PQ-CID) and described in U.S. Pat. No. 6,949,743 to Schwartz, may be employed in place of conventional CID by resonance excitation. In the PQD technique, the RF trapping voltage is increased prior to or during the period of kinetic excitation, and then reduced after a short delay period following termination of the excitation voltage in order to retain relatively low mass product ions in the trap. The PQD technique provides for more energetic collisional activation of target ions than does the original resonance excitation CID technique, while still retaining the lower mass product ions for subsequent analysis.
FIG. 1B schematically illustrates yet a third known method of providing collision induced dissociation. In this method, selected ions are temporarily stored in a multipole ion storage device 152, which may, for instance, comprise a quadrupole ion trap. At a certain time, an electrical potential on a gate electrode assembly 154 is changed so as to accelerate the selected ions 106 out of the ion storage device and into a collision cell 156 containing molecules 108 of an inert target gas. The ions are accelerated so as to collide with the target molecules at a kinetic energy that is determined by the difference in the potential offsets between the collision cell and the storage device. This method may be referred to by the acronyms HCD or HCID.
Photo-dissociation is another commonly employed fragmentation method in the field of mass spectrometry. For instance, in the technique known as Infrared Multiphoton Dissociation (IRMPD), infrared light from a laser is introduced into a vacuum chamber containing ions, such as an ion trap, so as to excite certain vibrational modes and thereby cause fragmentation. The IRMPD technique only works well under low pressure (high-vacuum) conditions. At higher pressures, ultraviolet light (for instance, from an ultraviolet lamp or a laser) can be used to excite electronic states within a molecule or ion, and thereby cause dissociation (or ionization). The infrared or UV light may be applied either continuously (that is, as a continuous wave) or else pulsed or chopped over a certain time period. Thus, in these photo-dissociation techniques, the power of the laser light or the energy per pulse is an important experimental variable as are, also, the light wavelength and the total time duration of exposure.
One remarkable aspect of the various ion fragmentation techniques is the fact that they are applicable to such a wide variety of precursors; masses, charges, shapes, and ion stabilities. However, to achieve the most efficient conversion of precursor ions to product ions, certain experimental parameters must be optimized, such as the collision energy, the target gas pressure, laser power or energy per pulse (for IRMPD and APPI) and possibly target gas constituents. Precursor ions of different size and structure have different internal energy requirements to maximize their unimolecular dissociation rates, and in general, collision energy must be increased as the mass of the precursor goes up and the charge of the precursor goes down. To maximize experimental throughput, a fragmentation energy dependence on mass and charge is typically calibrated and stored in the instrument, so that the appropriate parameters may be automatically varied in a data-dependent manner. The object of this disclosure is to provide an improved fragmentation energy calibration method that increases the likelihood that a given user-input fragmentation energy setting will appropriately fragment a precursor of a given mass and charge.